Benzo[1,2,5]oxadiazoles and benzol[1,2,5]thiadiazoles useful as histopathological staining agents, imaging agents and biomarkers

ABSTRACT

The present invention relates to novel benzo[1,2,5]oxadiazoles and benzo[1,2,5]thiadiazoles of formula (I) wherein X is O or S, R 1  is 5-(2-fluoro-ethylamino)-thiazol-2-yl, 5-(2- 18 F-ethylamino)-thiazol-2-yl or a group of formula (a) wherein Y is CH or N, R 2  is NHCH 3 , NH 11 CH 3 , N(CH 3 ) 11 CH 3 , N(CH 3 ) 2 , N( 11 CH 3 ) 2 , NH(CH 2 ) n F, NH(CH 2 ) n   18 F, N(CH 3 )(CH 2 ) n F, N(CH 3 )—(CH 2 ) n   18 F, O—(CH 2 ) n F, O—(CH 2 ) n   18 F, CONH(CH 2 )nF or CONH(CH 2 ) n   18 F (n being in each case 2 to 4) and R 3  is hydroxy, (C1-4)alkoxy, hydrogen or nitro, in free base or acid addition salt form; their preparation, their use as markers in diagnosis and compositions containing them.

The present invention relates to novel benzo[1,2,5]oxadiazoles andbenzo[1,2,5]thiadiazoles, their preparation, their use as markers andcompositions containing them.

More particularly the invention provides a compound of formula I

wherein X is O or S, R₁ is 5-(2-fluoro-ethylamino)-thiazol-2-yl,5-(2-¹⁸F-ethylamino)-thiazol-2-yl or a group of formula (a)

wherein Y is CH or N, R₂ is NHCH₃, NH¹¹CH₃, N(CH₃)¹¹CH₃, N(CH₃)₂,N(¹¹CH₃)₂, NH(CH₂)_(n)F, NH(CH₂)_(n) ¹⁸F, N(CH₃)—(CH₂)_(n)F,N(CH₃)—(CH₂)_(n) ¹⁸F, O—(CH₂)_(n)F, O—(CH₂)_(n) ¹⁸F, CONH(CH₂)_(n)F orCONH(CH₂)_(n) ¹⁸F (n being in each case 2 to 4) and R₃ is hydroxy,(C₁₋₄)alkoxy, hydrogen or nitro, in free base or acid addition saltform.

The term (C1-4)alkoxy as used herein relates to a radical comprising analkyl moiety with from and including 1 up to and including 4 C atoms andis linear or branched; preferably (C1-4)alkoxy is methoxy, ethoxy orn-propoxy.

Natural occurring carbon consists of the isotopes ¹²C (98.90%), ¹³C(1.10%) and traces of ¹⁴C. The term “¹¹C” denotes that a higher ratio ofthe respective carbon atoms in a radical or moiety correspond to ¹¹Cisotopes as compared to natural occurring carbon, especially a radicalor moiety wherein at least 25%, preferably at least 90%, more preferablyat least 95%, of the carbon atoms are ¹¹C isotopes.

The ¹¹C isotope can be prepared by methods known as such. Since thehalf-life period is short (about 20 min), the isotope needs to befreshly prepared shortly before usage in reagents by employment of acyclotron. The methylation reaction using ¹¹C-methyl iodide is the mostused reaction in ¹¹C labelling synthesis. A suitable method is based onthe reduction of trapped ¹¹C-carbon dioxide with lithium aluminumhydridein tetrahydrofuran, followed by removal of solvent. Hydroiodic acid isadded and the formed ¹¹C-methyl iodide distilled in a stream of nitrogengas to the reaction flask.

Natural occurring fluorine consists of the isotope ¹⁹F (100%). The term“¹⁸F” denotes that a higher ratio of the respective fluorine atoms in aradical or moiety correspond to ¹⁸F isotope as compared to naturaloccurring fluorine, especially a radical or moiety wherein at least 25%,preferably at least 90%, more preferably at least 95%, of the fluorineatoms are ¹⁸F isotopes.

The ¹⁸F isotope can be prepared by methods known as such. Since thehalf-life period is short (about 110 min), the isotope needs to befreshly prepared shortly before usage in reagents by employment of acyclotron. ¹⁸F can be obtained, e.g., by irradiating a target containing¹⁸O—H₂O with a proton beam, in a cyclotron. In this process, ¹⁸F isobtained from the ¹⁸O(p,n) ¹⁸F nuclear reaction and used immediately togenerate final radiomarkers as described, e.g. in “Fundamentals ofPositron Emission tomography and Applications in Preclinical DrugDevelopment”, Simon R Cherry, J Clin Pharmacol 2001; 41:482-491, and thereferences cited therein, which publication is included in the presentpatent filing by reference.

For diagnostic purposes, those compounds of formula I are preferredcomprising ¹¹C or ¹⁸F.

In a further aspect, the invention provides a process for the productionof the compounds of formula I and their salts, comprising the steps of

-   a) for the production of a compound of formula I which contains no    ¹¹C or ¹⁸F atom, reacting a compound of formula II

wherein X is as defined above and Hal is Cl, Br or I, with5-(2-fluoro-ethylamino)thiazolyl-2-boronic acid or a compound of formulaIII

wherein Y and R₃ are as defined above and R′₂ is a group R₂ as definedabove which contains no ¹¹C or ¹⁸F atom, or

-   b) for the production of a compound of formula I wherein R₁ is    5-(2¹⁸F-ethylamino)-thiazol-2-yl, reacting a compound of formula I    wherein R₁ is 5-(2-mesyloxy-ethylamino)-thiazol-2-yl or    5-(2-tosyloxy-ethylamino)-thiazol-2-yl with ¹⁸FΘ, or-   c) for the production of a compound of formula I wherein R₂ is    NH¹¹CH₃, N(CH₃)¹¹CH₃ or N(¹¹CH₃)₂, reacting a compound of formula I    wherein R₂ is NH₂ or NHCH₃ with ¹¹CH₃I, or-   d) for the production of a compound of formula I wherein R₂ is    NH(CH₂)_(n) ¹⁸F, N(CH₃)—(CH₂)_(n) ¹⁸F, O—(CH₂)_(n) ¹⁸F or    CONH(CH₂)_(n) ¹⁸F, reacting a compound of formula I wherein R₂ is,    respectively, NH(CH₂)_(n)OTs or NH(CH₂)_(n)OMs, N(CH₃)—(CH₂)_(n)OTs    or N(CH₃)—(CH₂)_(n)—OMs, O—(CH₂)_(n)OTs or O—(CH₂)_(n)—OMs, or    CONH(CH₂)_(n)OTs or CONH(CH₂)_(n)OMS, with ¹⁸FΘ,    and recovering the resulting compound of formula I in free base form    or in form of an acid addition salt.

The reaction described under a) can be effected according to knownmethods, for example as described in Example 1.

Alternatively to process a), conventional methods can be used for theproduction of compounds of formula I which contain no ¹¹C or ¹⁸F atom,for example as described in Examples 4, 7 and 13 to 25.

The reactions described under b), c) and d) can be effected according toconventional methods.

Working up the reaction mixtures and purification of the compounds thusobtained may be carried out in accordance to known procedures.

Acid addition salts may be produced from the free bases in known manner,and vice-versa.

The agents of the invention have a high affinity to β-amyloid deposits,especially extracellular β-amyloid deposits, and bind to these depositsby means of physical or chemical interaction.

Hence, the agents of the invention can be used in the therapy ordiagnosis of diseases wherein such β-amyloid deposits are observed, suchas Alzheimer's Disease. Preferably, the agents of the invention are usedin diagnosis.

In particular, compounds of formula I in free base or acid addition saltform, hereinafter referred to as agents of the invention, exhibitvaluable properties as histopathological staining agents, imaging agentsand/or biomarkers, hereinafter “markers”.

More particularly, the agents of the invention are useful as markers forlabeling pathological structures such as extracellular β-amyloiddeposits, e.g. in the brain of patients with Alzheimer's disease (seeExample 26).

The agents of the invention are therefore useful for the early diagnosisand prevention of Alzheimer's disease and for monitoring theeffectiveness of therapeutic treatments of Alzheimer's disease.

The advantages of assessing amyloid deposits in vivo and non-invasivelyusing markers capable of labeling these structures have been reportede.g. in WO 00/10614, which is incorporated herein by reference.

In accordance with the above, the present invention provides acomposition for labeling histopathological structures in vivo or invitro, comprising an agent of the invention.

In a further aspect, the present invention provides a method forlabeling histopathological structures in vitro or in vivo, whichcomprises contacting brain tissue with an agent of the invention.

Said brain tissue comprises for example β-amyloid deposits.

Contacting the brain tissue with the agent of the invention is forexample effected by administering the agent of the invention to apatient, e.g. a patient with Alzheimer's disease.

The method of the invention may comprise a further step aimed atdetermining whether the agent of the invention labeled the targetstructure.

If the agent of the invention is a non-radioactive compound of formulaI, said further step may be effected by observing the target structureusing fluorescence microscopy.

If the agent of the invention is a radioactive compound of formula I,said further step may be effected by observing the target structureusing positron emission tomography (PET).

Labeling histopathological structures in vitro is effected, for example,for detecting histopathological hallmarks of Alzheimer's disease.

Labeling histopathological structures in vivo is effected, for example,for diagnosing Alzheimer's disease in a patient or for monitoring theeffectiveness of a therapeutic treatment of Alzheimer's disease.

In further aspects, the present invention relates to

-   -   the use of a compound of formula I for the manufacture of a        preparation for the treatment or diagnosis of Alzheimer's        disease;    -   a package comprising a compound of formula I wherein R₂ is NH₂        or NHCH₃ together with instructions for the production of a        compound of formula I wherein R₂ is NH¹¹CH₃, N(CH₃)¹¹CH₃ or        N(¹¹CH₃)₂ by reaction of the starting material with freshly        prepared ¹¹CH₃I; and to    -   a package comprising as starting material a compound of formula        I wherein R₂ is NH(CH₂)_(n)OTs, NH(CH₂) _(n)OMs, N(CH₃)—(CH₂)        _(n)OTs, N(CH₃)—(CH₂) _(n)OMs, O—(CH₂) _(n)OTs, O—(CH₂)        _(n)—OMs, CONH(CH₂) _(n)OTs or ONH(CH₂) _(n)OMs, wherein OMs        corresponds to mesylate and OTs to tosylate, together with        instructions for the production of a compound of formula I        wherein R₂ is NH(CH₂)_(n) ⁸F, N(CH₃)—(CH₂) _(n) ¹⁸F, O—(CH₂)        _(n) ¹⁸F or CONH(CH₂)_(n) ¹⁸F by a suitable reaction cascade of        the starting material with ¹⁸FΘ.

The following Examples illustrate the invention.

Abbreviations

-   Boc t-butoxycarbonyl-   DMAP dimethylamino pyridine-   DMF dimethyl formamide-   DMSO dimethyl sulfoxide-   Dppf 1,1′-bis(diphenylphosphino)ferrocene-   EDC N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide-   Eq. equivalents-   HOBt 1-hydroxybenzotriazole-   HPLC high pressure liquid chromatography-   NaHMDS sodium hexamethyldisilazane-   NMR nuclear magnetic resonance-   OMs mesylate-   OTs tosylate-   THF tetrahydrofurane

EXAMPLE 1 (4-Benzo[1,2,5]oxadiazol-5-yl-phenyl)-dimethyl-amine

500 mg (2.5 mmol) 5-bromo-benzo[1,2,5]oxadiazole and 313 mg (0.1 eq.)tetrakis-(triphenylphosphine)palladium are stirred for 30 minutes atroom temperature in 10 mL 1,2-dimethoxyethane. 533 mg (2 eq.) sodiumcarbonate are dissolved in 1.8 mL water and added to the reactionmixture, followed by 663 mg (1.6 eq.) 4-(dimethylamino)phenylboronicacid. After stirring at reflux for 18 h, the reaction mixture is cooledto room temperature and extracted with ethyl acetate and water. Theorganic phases are combined, dried with magnesium sulfate, filtered andevaporated. The residue is column chromatographed (hexane, thenhexane/ethyl acetate 8:1 then 5:1) to yield the desired product as ayellow solid. MS(ES+): 240 (M+1).

¹H-NMR (CDCl₃, 400 MHz): delta (ppm)=7.83 (d, H_(arom)); 7.82 (s,H_(arom)); 7.73 (d, H_(arom)); 7.58, 6.81 (2d, 2×2H_(arom)); 3.05 (s,2Me).

The following compounds of examples 2 to 6 can be prepared according tothe same procedure:

EXAMPLE 2 (4-Benzo[1,2,5]thiadiazol-4-yl-phenyl)-dimethyl-amine

Starting from 4-iodo-benzo[1,2,5]thiadiazole and4-(dimethylamino)phenylboronic acid. Orange powder. MS(ES+): 256 (M+1).

EXAMPLE 3 (4-Benzo[1,2,5]oxadiazol-4-yl-phenyl)-dimethyl-amine

Starting from 4-chloro-benzo[1,2,5]oxadiazole and4-(dimethylamino)phenylboronic acid. Orange powder. MS(ES+): 240 (M+1).

EXAMPLE 4 (3Benzo[1,2,5]oxadiazol-5-yl-phenyl)-methyl-amine

Starting from 5-benzo[1,2,5]oxadiazoleboronic acid and(3-bromo-phenyl)-methyl-amine. Yellow powder. MS(ES+): 226 (M+1).

EXAMPLE 5 5-[4-(2-Fluoro-ethoxy)-phenyl]-benzo[1,2,5]oxadiazole

Starting from 5-benzo[1,2,5]oxadiazoleboronic acid and1-bromo-4-(2-fluoro-ethoxy)-benzene. Yellow-beige powder. MS(ES+): 259(M+1).

EXAMPLE 6 (3-Benzo[1,2,5]oxadiazol-5-yl-phenyl)-dimethyl-amine

Starting from 5-benzo[1,2,5]oxadiazoleboronic acid and(3-bromo-phenyl)-dimethyl-amine. Orange-yellow powder. MS(ES+): 240(M+1).

EXAMPLE 7 (2Benzo[1,2,5]oxadiazol-5-yl-phenyl)-dimethyl-amine

523 mg (1.65 eq.) bis(pinacolato)diboran, 552 mg (4.5 eq.) potassiumacetate and 51 mg (0.01 eq.) PdCl(dppf)-CH₂Cl₂ are added to a solutionof 373 mg 5-bromo-benzo[1,2,5]oxadiazole in 8 mL DMF and heated to 80°C. under continuous stirring for 6 hours. 662 mg (5 eq.) sodiumcarbonate in 3.3 mL water are added together with 500 mg (1 eq.)2-bromo-N,N-dimethylaniline and 51 mg (0.01 eq.) PdCl(dppf)-CH₂Cl₂ tothe reaction mixture, which is stirred for another 18 hours at 80° C.,cooled to room temperature and extracted with ethyl acetate and water.The combined organic phases are washed with brine, dried over magnesiumsulfate and evaporated. The residue is column chromatographed (hexane,then hexane/ethyl acetate 8:1) to yield the desired product as an orangeresin. MS(ES+): 240 (M+1).

¹H-NMR (CDCl₃, 400 MHz): delta (ppm)=7.77 (s, H_(arom)); 7.71, 7.69 (2d,2H_(arom)); 7.25, 7.03 (2m, 2×2H_(arom)); 2.53 (s, 2Me).

The following compounds of Examples 8 to 12 are synthesized according tothe same procedure:

EXAMPLE 8 (2Benzo[1,2,5]thiadiazol-5-yl-phenyl)-dimethyl-amine

Starting from 5-bromo-benzo[1,2,5]thiadiazole and2-bromo-N,N-dimethylaniline. Orange resin. MS(ES+): 256 (M+1).

EXAMPLE 9 (3Benzo[1,2,5]oxadiazol-4-yl-phenyl)-methyl-amine

Starting from 5-chloro-benzo[1,2,5]oxadiazole and(3-bromo-phenyl)-methyl-amine. Yellow powder. MS(ES+): 226 (M+1).

The starting material is obtained as follows:

(3-Bromo-phenyl)-methyl-amine

2 g (11.6 mmol) 3-Bromoaniline is dissolved in 15 mL DMF and treatedwith 716 mg (1.1 eq.) KOH and 0.722 mL (1 eq.) iodomethane undercontinuous stirring at room temperature for 20 hours. The reactionmixture is extracted with ethyl acetate and water, the combined organicphases are washed with brine and dried with sodium sulfate andevaporated to dryness. The residue is column chromatographed (ethylacetate/petroleum ether 2:5) to yield after evaporation the desiredproduct as a yellow oil.

EXAMPLE 10 4-[4-(2-Fluoro-ethoxy)-phenyl]-Benzo[1,2,5]oxadiazole

Starting from 4-chloro-benzo[1,2,5]oxadiazole and1-bromo-4-(2-fluoro-ethoxy)-benzene. Yellow crystals. MS(ES+): 259(M+1).

EXAMPLE 11 5Benzo[1,2,5]oxadiazol-5-yl-N-(2-fluoro-ethyl)-nicotinamide

Starting from 5-bromo-benzo[1,2,5]oxadiazole and5-bromo-N-(2-fluoro-ethyl)-nicotinamide. Beige powder. MS(ES−): 285(M−1).

The starting material is obtained as follows:

5-Bromo-N-(2-fluoro-ethyl)-nicotinamide

2.03 g (10 mmol) 5-bromonicotinic acid, 1 g (1 eq.) 2-fluoroethylamine,2.31 g (1.2 eq.) EDC.HCl, 123 mg (0.1 eq.) DMAP and 5.6 mL (4 eq.)triethylamine are stirred in 60 mL dichloromethane for 20 hours at roomtemperature. The reaction mixture is extracted with dichloromethane andwater, the combined organic phases are washed with brine, dried withsodium sulfate and evaporated. The residue is triturated in diethyletherand hexane to yield after filtration the desired product as a whitepowder. MS(ES+): 347 and 349 (M+1).

EXAMPLE 12 4Benzo[1,2,5]oxadiazol-5-yl-N-(2-fluoro-ethyl)-nicotinamide

Starting from 4-chloro-benzo[1,2,5]oxadiazole and5-bromo-N-(2-fluoro-ethyl)-nicotinamide. Beige powder. MS(ES−): 285(M−1).

EXAMPLE 13 (4Benzo[1,2,5]oxadiazol-5-yl-phenyl)-methyl-amine

510 mg (1.57 eq.) (4-benzo[1,2,5]oxadiazol-5-yl-phenyl)-methyl-carbamicacid tert-butyl ester are dissolved in 10 mL dichloromethane, treatedwith 0.126 mL (1.05 eq.) trifluoroacetic acid and stirred overnight atroom temperature. The reaction mixture is evaporated and the residuecolumn chromatographed (hexane, hexane/ethyl acetate 4:1 then 3:1) toyield the desired product as an orange solid. MS(ES+): 226 (M+1).

¹H-NMR (CDCl₃, 400 MHz): delta (ppm)=7.82 (d, H_(arom)); 7.81 (s,H_(arom)), 7.71 (d, H_(arom)); 7.54, 6.72 (2d, 2×2H_(arom)); 2.92 (s,Me).

The starting material is obtained according to the procedure describedin Example 7 from 5-bromo-benzo[1,2,5]oxadiazole and(4-bromo-phenyl)-methyl-carbamic acid tert-butyl ester, as a yellowpowder.

¹H-NMR (CDCl₃, 400 MHz): delta (ppm)=7.84 (s, H_(arom)); 7.82, 7.63 (2d,2H_(arom)); 7.54, 7.18 (2d, 2×2 H_(arom)); 3.26 (s, Me); 1.43 (s, tBu).

The following compounds of example 14 to 16 are prepared according tothe same procedure:

EXAMPLE 14 (4Benzo[1,2,5]thiadiazol-5-yl-phenyl)-methyl-amine

Starting from (4-benzo[1,2,5]thiadiazol-5-yl-phenyl)-methyl-carbamicacid tert-butyl ester. MS(ES+): 242 (M+1).

The starting compound is obtained according to the procedure describedin Example 7 from 5-bromo-benzo[1,2,5]thiadiazole and(4-bromo-phenyl)-methyl-carbamic acid tert-butyl ester, as a yellowpowder.

¹H-NMR (CDCl₃, 400 MHz): delta (ppm)=8.08 (s, H_(arom)); 7.98, 7.80 (2d,2H_(arom)); 7.60, 7.33 (2d, 2×2 H_(arom)); 3.26 (s, Me); 1.43 (s, tBu).

The starting material is obtained as follows:

a) (4-Bromo-phenyl)-methyl-carbamic acid tert-butyl ester

0.263 mL (1.1 eq.) iodomethane are added dropwise at room temperature toa stirred suspension of 1.1 g (4 mmol) (4-bromo-phenyl)-carbamic acidtert-butyl ester and 2 g (1.6 eq.) cesium carbonate in 15 mL DMF. Afterstirring for an additional 18 hours, the reaction mixture is extractedwith water/ethyl acetate and the combined organic phases are washed withbrine, dried over magnesium sulfate and evaporated to yield the desiredproduct as a colorless oil, which is used without further purification.

b) (4-Bromo-phenyl)-carbamic acid tert-butyl ester

2 mL (1.2 eq.) Triethylamine are added to a solution of 2 g (11.6 mmol)p-bromo-aniline in 25 mL THF. The reaction mixture is cooled to 0° C.and treated with 2.66 g (1.05 eq.) Boc₂O under continuous stirring, thenallowed to reach 20° C., stirred for an additional 20 hours andextracted with water/ethyl acetate. The combined organic phases arewashed with brine, dried over magnesium sulfate and evaporated. Theresidue is column chromatographed (hexane, hexane/ethyl acetate 7:1 then4:1) to yield after evaporation and recrystallization from hexane thedesired product as a white powder.

EXAMPLE 15 (4Benzo[1,2,5]oxadiazol-5-yl-3-methoxy-phenyl)-methyl-amine

Starting from(4-Benzo[1,2,5]oxadiazol-5-yl-3-methoxy-phenyl)-methyl-carbamic acidtert-butyl ester.

¹H-NMR (CD₃OD, 400 MHz): delta (ppm)=7.78 (m, 3H_(arom)); 7.25 (d,H_(arom)); 6.38 (m, H_(arom)); 6.36 (m, H_(arom)); 3.83 (s, Me); 2.84(s, Me).

The starting compound is obtained according to the procedure describedin Example 7 from 5-bromo-benzo[1,2,5]oxadiazole and(4-bromo-3-methoxy-phenyl)-methyl-carbamic acid tert-butyl ester, as ayellow powder. MS(ES+): 378 (M+23).

The starting material is obtained as follows:

(4-Bromo-3-methoxy-phenyl)-methyl-carbamic acid tert-butyl ester

0.5 mL (1.1 eq.) iodomethane are added dropwise at room temperature to astirred suspension of 2 g (6.6 mmol)(4-benzo[1,2,5]oxadiazol-5-yl-3-methoxy-phenyl)-methyl-carbamic acidtert-butyl ester and 3.2 g (1.5 eq.) cesium carbonate in 50 mL DMF.After stirring for an additional 17 hours, the reaction mixture isextracted with water/ethyl acetate and the combined organic phases arewashed with brine, dried over magnesium sulfate and evaporated to yieldthe desired product as a yellow oil, which is used without furtherpurification.

EXAMPLE 16(4Benzo[1,2,5]oxadiazol-5-yl-3-methoxy-phenyl)-(2-fluoro-ethyl)-amine

Starting from(4-benzo[1,2,5]oxadiazol-5-yl-3-methoxy-phenyl)-(2-fluoro-ethyl)-carbamicacid tert-butyl ester.

¹H-NMR (CD₃OD, 400 MHz): delta (ppm)=7.78-7.84 (m, 3H_(arom)); 7.26 (d,H_(arom)); 6.44 (s, H_(arom)); 6.40 (d, H_(arom)); 4.68 (t, CH ²F); 4.58(t, CH ₂F); 3.82 (s, Me); 3.55 (t, CH ₂N); 3.46 (t, CH ₂N).

The starting compound is obtained according to the procedure describedin Example 7 from 5-bromo-benzo[1,2,5]oxadiazole and(4-bromo-3-methoxy-phenyl)-(2-fluoro-ethyl)-carbamic acid tert-butylester, as a yellow glass. MS(ES+): 410 (M+23).

The starting material is obtained as follows:

(4-Bromo-3-methoxy-phenyl)-methyl-carbamic acid tert-butyl ester

0.63 mL (1.25 eq.) 1-bromo-2-fluoroethane are added at room temperatureto a stirred suspension of 2 g (6.6 mmol)(4-benzo[1,2,5]oxadiazol-5-yl-3-methoxy-phenyl)-methyl-carbamic acidtert-butyl ester and 3.2 g (1.5 eq.) cesium carbonate in 50 mL DMF.After stirring for 12 hours at 50 to 55° C., the reaction mixture iscooled to rt. And extracted with water/ethyl acetate. The combinedorganic phases are washed with brine, dried over magnesium sulfate andevaporated to yield the desired product as a yellow oil, which is usedwithout further purification.

EXAMPLE 17(4Benzo[1,2,5]oxadiazol-5-yl-phenyl)-(2-fluoro-ethylmethyl-amine

220 mg (0.98 mmol) (4-benzo[1,2,5]oxadiazol-5-yl-phenyl)-methyl-amineare dissolved in 5 mL DMF, treated with 1.9 g (6 eq.) cesium carbonate,320 mg (2 eq.) potassium iodide and 0.364 mL (5 eq.)1-bromo-2-fluoroethane, and stirred for 20 hours at 50° C. The reactionmixture is extracted with water and ethyl acetate, the combined organicphases are washed with water and brine, dried over magnesium sulfate andevaporated. The residue is column chromatographed (gradient from hexaneto ethyl acetate) to yield the desired product as an orange powder.

¹H-NMR (CDCl₃, 400 MHz): delta (ppm)=7.77 (d, H_(arom)); 7.75 (s,H_(arom)); 7.65 (d, H_(arom)); 7.51, 6.75 (2d, 2×2H_(arom)); 4.57, 3.67(2m, 2CH₂); 3.04 (s, Me).

EXAMPLE 18 5-[3-(2-Fluoroethoxy)-phenyl]-Benzo[1,2,5]oxadiazole

600 mg (2.83 mmol) 3-benzo[1,2,5]oxadiazol-5-yl-phenol are dissolved in15 mL DMF and stirred for 20 hours at room temperature in the presenceof 1.15 g (1.25 eq.) cesium carbonate and 0.264 mL1-bromo-2-fluoroethane. The reaction mixture is extracted with water andethyl acetate, the combined organic phases are washed with water andbrine, dried with magnesium sulfate and evaporated to dryness. Theresidue is column chromatographed (gradient from hexane to hexane/ethylacetate 3:2) to yield the desired product as a pale yellow powder.MS(ES+): 259 (M+1).

¹H-NMR (CDCl₃, 400 MHz): delta (ppm)=7.95 (s, H_(arom)); 7.90 (d,H_(arom)); 7.70 (d, H_(arom)); 7.42 (m, H_(arom)); 7.27 (m, H_(arom));7.22 (s, H_(arom)); 7.04 (m, H_(arom)); 4.80, 4.31 (2m, 2CH₂).

The starting material 3-benzo[1,2,5]oxadiazol-5-yl-phenol is obtainedaccording to the procedure described in Example 1 starting from5-bromo-benzo[1,2,5]oxadiazole and 3-hydroxyphenylboronic acid, as alight yellow powder.

¹H-NMR (CDCl₃, 400 MHz): delta (ppm)=7.94 (s, H_(arom)); 7.92, 7.68 (2d,2 H_(arom)); 7.38 (t, H_(arom)); 7.24 (d, H_(arom)); 7.13 (s, H_(arom));6.94 (d, H_(arom)); 4.93 (OH).

EXAMPLE 19(2Benzo[1,2,5]oxadiazol-5-yl-thiazol-5-yl)-(2-fluoro-ethyl)-amine

53 mg (0.2 mmol) benzo[1,2,5]oxadiazole-5-carboxylic acid[(2-fluoro-ethylcarbamoyl)-methyl]-amide and 97 mg (1.2 eq.) Lawesson'sreagent are suspended in 20 mL toluene and heated to reflux for onehour. After cooling to room temperature, the reaction mixture is pouredonto ice-water containing 2 mL of a saturated aqueous solution of sodiumcarbonate, then extracted with ethyl acetate, The combined organicphases are dried with sodium sulfate and evaporated to dryness. Theresidue is column chromatographed (petroleum ether/ethyl acetate 3:2) toyield after evaporation of the corresponding fractions the desiredproduct as an orange powder. MS (ES+): 265 (M+1).

¹H-NMR (CDCl₃, 400 MHz): delta (ppm)=8.12 (d, H_(arom)); 7.95 (s,H_(arom)); 7.82 (d, H_(arom)); 7.04 (s, H_(het)); 4.70, 3.53 (2m, 2CH₂).

The starting materials were obtained as follows:

a) Benzo[1,2,5]oxadiazole-5-carboxylic acid[(2-fluoro-ethylcarbamoyl)-methyl]-amide

221 mg (1 mmol) [(benzo[1,2,5]oxadiazole-5-carbonyl)-amino]-acetic acid,120 mg (1.2 eq.) 2-fluoroethylamine, 162 mg (1.2 eq.) HOBt and 230 mg(1.2 eq.) EDC.HCl are dissolved in 18 mL DMF and cooled to 5° C. 0.7 mL(4 eq.) Hünig's base are added under stirring, the reaction mixture isstirred for an additional 3 hours at room temperature then extractedwith water and ethyl acetate. The combined organic phases are washedwith brine, dried with sodium sulfate and evaporated to yield thedesired product as a beige solid. MS (ES+): 267 (M+1).

b) [(Benzo[1,2,5]oxadiazole-5-carbonyl)-amino]-acetic acid

500 mg (2.13 mmol) [(benzo[1,2,5]oxadiazole-5-carbonyl)-amino]-aceticacid methyl ester are treated in 50 mL methanol with 6 mL (3 eq.) 1N KOHin methanol containing 10% water at room temperature for two hours, thencooled below 5° C. and neutralized with 6 mL 1 N aqueous HCl. Thereaction mixture is extracted with ethyl acetate, the combined organicphases are washed with brine and dried with sodium sulfate, thenevaporated to dryness to yield the desired product as a beige-pinkishpowder. MS (ES−): 220 (M−1)

c) [(Benzo[1,2,5]oxadiazole-5-carbonyl)-amino]-acetic acid methyl ester

492 mg (3 mmol) benzo[1,2,5]oxadiazole-5-carboxylic acid 376 mg (1 eq.)glycine methyl ester hydrochloride, 486 mg (1.2 eq.) HOBt and 690 mg(1.2 eq.) EDC.HCl are dissolved in 20 mL DMF under argon and thereaction mixture is stirred at room temperature for 90 minutes afteraddition of 2 mL (4 eq.) Hünig's base. The reaction mixture is pouredonto ice and brine, and extracted with ethyl acetate. The combinedorganic phases are dried with sodium sulfate and evaporated to yield thedesired product as a beige powder. MS (ES+): 236 (M+1).

EXAMPLE 20 (4Benzo[1,2,5]oxadiazol-5-yl-phenyl)-(2-fluoro-ethyl)-amine

391 mg (1 mmol)(4-benzo[1,2,5]oxadiazol-5-yl-phenyl)-(2-fluoro-ethyl)-carbamic acidbenzyl ester are hydrogenated in the presence of Pd/C, the reactionmixture filtered through celite and evaporated to dryness. The residuesis column chromatographed (dichloromethane/petroleum ether 8:2) to yieldthe desired product as a yellow powder. MS(ES+): 258 (M+1).

¹H-NMR (CDCl₃, 400 MHz): delta (ppm)=7.82 (d, H_(arom)); 7.81 (s,H_(arom)); 7.70 (d, H_(arom)); 7.53 (m, 2H_(arom)); 6.75 (m, 2H_(arom));4.66, 3.51 (2m, 2CH₂).

The starting material is obtained according to the procedure describedin Example 1 from 5-benzo[1,2,5]oxadiazoleboronic acid and(4-bromo-phenyl)-(2-fluoro-ethyl)-carbamic acid benzyl ester, as a lightbrown resin. MS(ES+): 392 (M+1).

The starting compound can be synthesized as follows:

a) (4-bromo-phenyl)-(2-fluoro-ethyl)-carbamic acid benzyl ester

2.9 g (6 mmol) toluene-4-methanesulfonic acid2-[benzyloxycarbonyl-(4-bromo-phenyl)-amino]-ethyl ester are dissolvedin 100 mL THF and stirred for 18 hours at room temperature afteraddition of a 1 M solution of tetrabutyl-ammonium fluoride in THF. Thereaction mixture is poured onto ice and extracted with tertbutyl-methylether and a saturated aqueous solution of sodium bicarbonate. Thecombined organic phases are washed with brine, dried with sodium sulfateand evaporated to dryness. The crude product is column chromatographed(petroleum ether/ethyl acetate 4:1) to yield 920 mg desired product as ayellow resin. MS(ES+): 352 (M+1).

b) Toluene-4-methanesulfonic acid2-[benzyloxycarbonyl-(4-bromo-phenyl)-amino]-ethyl ester

1.3 g (6 mmol) 2-(4-bromo-phenylamino)-ethanol are dissolved in 150 mLtoluene, treated with 0.93 mL (1.1 eq.) benzyloxycarbonylchloride and6.3 mL (1.05 eq.) 1N aqueous sodium hydroxide solution. The reactionmixture is stirred at room temperature for 18 hours, then the aqueousphase separated and extracted with ethyl acetate and water. The combinedorganic phases are washed with water and brine, then evaporated todryness to yield 2.1 g crude (4-bromo-phenyl)-(2-hydroxy-ethyl)-carbamicacid benzyl ester, which is reacted further without additionalpurification by dissolution in 100 mL dichloromethane, addition of 5 mL(excess) pyridine, cooling to 5° C. and dropwise addition in 10 minutesof 2.45 g p-toluenesulfonic acid anhydride in 20 mL dichloromethane. Thereaction mixture is stirred an additional 2 hours at room temperatureand poured onto ice, then extracted at 10° C. with dichloromethane andan aqueous solution of sodium bicarbonate. The combined organic phasesare dried with sodium sulfate and evaporated to yield the desiredproduct as a light brownish resin, which is used without furtherpurification.

EXAMPLE 21 (4Benzo[1,2,5]oxadiazol-5-yl-2-nitro-phenyl)-methyl-amine

140 mg (0.62 mmol) (4-benzo[1,2,5]oxadiazol-5-yl-phenyl)-methyl-amineare stirred at 0° C. in 2 mL concentrated sulfuric acid, then treatedwith 66 mg (1.05 eq.) potassium nitrate and stirred an additional 4hours. The reaction mixture is poured onto ice, the precipitate filteredoff, thoroughly washed with water and dried under high vacuum. The crudeproduct is column chromatographed with ethyl acetate/petroleum ether 1:2to yield after evaporation of the product-containing fraction thedesired product as an orange solid. MS(ES−): 269 (M−1).

¹H-NMR (CDCl₃, 400 MHz): delta (ppm)=8.55 (s, H_(arom)); 8.22 (br.s,NH); 7.92 (d, H_(arom)); 7.91 (s, H_(arom)); 7.82 (d, H_(arom)); 7.72(d, H_(arom)); 7.01 (d, H_(arom)); 3.13 (s, Me).

EXAMPLE 22 2Benzo[1,2,5]oxadiazol-5-yl-5-methylamino-phenol

100 mg (0.4 mmol) of(4-benzo[1,2,5]oxadiazol-5-yl-3-methoxy-phenyl)-methyl-amine are stirredat −10 to 0° C. in 1.3 mL of CH₂Cl₂, then over 5 minutes treated with1.3 mL AlBr₃ (1.0 M solution in CH₂Br₂, 3 eq.) and stirred another 17 hunder warming to rt. Then, the mixture is poured onto brine/ethylacetate and the pH adjusted to 6 using NaHCO₃. Then the aqueous phase isextracted with ethyl acetate, the combined organic layers dried withsodium sulfate and concentrated. The crude product is columnchromatographed with ethyl acetate/hexanes 1:1 to yield afterevaporation of the product-containing fractions the desired product as ayellow solid.

¹H-NMR (CD₃OD, 400 MHz): delta (ppm)=7.88-7.94 (m, 2H_(arom)); 7.79 (d,H_(arom)); 7.24 (d, H_(arom)); 6.29 (d, H_(arom)); 6.21 (m, H_(arom));2.81 (s, Me).

EXAMPLE 23 2Benzo[1,2,5]oxadiazol-5-yl-5-(2-fluoro-ethylamino)-phenol

10 mg (0.03 mmol) of(4-benzo[1,2,5]oxadiazol-5-yl-3-methoxy-phenyl)-(2-fluoro-ethyl)-amineare dissolved in 0.3 mL acetic acid and 0.3 mL hydrobromic acid (33% inacetic acid) and stirred at 80 QC for 72 h. Then the solvents areremoved. The mixture of starting material and desired product can beseparated by HPLC (20 to 100% CH₃CN/H₂O (6′), 100% CH₃CN (1.5°), 100 to20% CH₃CN/H₂O (0.5°): product elutes after 4.5°). MS(ES+): 274 (M+1).

EXAMPLE 24 (4Benzo[1,2,5]oxadiazol-5-yl-3-methoxy-phenyl)-dimethyl-amine

200 mg (0.8 mmol) of(4-benzo[1,2,5]oxadiazol-5-yl-3-methoxy-phenyl)-methyl-amine are stirredat 0 to 5° C. in 5 mL of DMF, then over 1 minute treated with 1 mLNaHMDS-solution (1.0 M, 1.25 eq.) and stirred another minute. Then, 0.5mL (7.8 mmol, 10 eq.) of methyliodide is added and stirred 2 minutesunder warming to rt. Then, the mixture is extracted with ethylacetate/aqueous NaHCO₃, the combined organic layers dried with sodiumsulfate and concentrated. This yields the desired product as areddish-yellow solid.

¹H-NMR (CD₃OD, 400 MHz): delta (ppm)=7.80-7.84 (m, 2H_(arom)); 7.36 (d,H_(arom)); 6.50 (d, H_(arom)); 6.45 (m, H_(arom)); 3.90 (s, MeO); 3.08(s, 2Me).

EXAMPLE 25 2Benzo[1,2,5]oxadiazol-5-yl-5-dimethylamino-phenol

140 mg (0.52 mmol) of(4-benzo[1,2,5]oxadiazol-5-yl-3-methoxy-phenyl)-dimethyl-amine arestirred at −10 to 0° C. in 1.7 mL of CH₂Cl₂, then over 5 minutes treatedwith 1.7 mL AlBr₃ (1.0 M solution in CH₂Br₂, 3.3 eq.) and stirredanother 19 h under warming to rt. Then, the mixture is extracted withbrine/ethyl acetate, the combined organic layers dried with sodiumsulfate and concentrated. The crude product is column chromatographedwith ethyl acetate/hexanes 1:4 to yield after evaporation of theproduct-containing fractions the desired product as a red solid.

¹H-NMR (CD₃OD, 400 MHz): delta (ppm)=7.90-7.94 (m, 2H_(arom)); 7.80 (d,H_(arom)); 6.43 (d, H_(arom)); 6.38 (m, H_(arom)); 4.00 (s, 2Me).

EXAMPLE 26

Staining of APP23 Mouse and Human ALZHEIMER Disease (AD) Brain SectionsUsing an Agent of the Invention or Thioflavine S.

Four-micrometer thick paraffin sections from an APP23 mouse at 26 monthsof age are deparaffinized in xylene and rehydrated. 10 mg of thecompound are dissolved in 1 mL DMSO and diluted with deionized water1:10. This staining solution is applied on sections for about 20 min.Section background is cleared by washing with 95% ethanol. Finallysections are dehydrated in 99% ethanol, cleared in xylene and mountedwith Vectashield™. Sections are investigated using fluorescencemicroscopy with the following filter combination: Excitation 450490 nm,emission 510 nm. Twenty micrometer thick cryotom sections from a ADbrain cortex are air dried and fixated in 4% PFA for 5 min. Afterwashing in tap water sections are stained either with Thioflavine S orwith the compound for 5 min and further processed as described above.The compound is dissolved in DMSO and diluted to a final concentrationof 0.01% with 50% Ethanol, Thioflavine S is dissolved in 50% Ethanol,final concentration is 0.01%.

Results:

(These results do not apply to the compounds of Examples 4, 5, 11, 12and 21, which are not fluorescent)

1) Staining of APP23 Mice Brain Sections

The agents of the invention strongly stain amyloid deposits and vasculardeposits in brain sections of APP23 mice.

2) Staining of Human AD Brain Sections:

Brain sections taken from frontal cortex of AD patients are stained withthe agents of the invention, and the results compared with a ThioflavineS stain. The agents of the invention intensely and selectively stainamyloid deposits.

1. A compound of formula I

wherein X is O or S, R₁ is 5-(2-fluoro-ethylamino)-thiazol-2-yl,5-(2-¹⁸F-ethylamino)-thiazol-2-yl or a group of formula (a)

wherein Y is CH or N, R₂ is NHCH₃, NH¹¹CH₃, N(CH₃)¹¹CH₃, N(CH₃)₂,N(¹¹CH₃)₂, NH(CH₂)_(n)F, NH(CH₂)_(n) ¹⁸F, N(CH₃)—(CH₂)_(n)F,N(CH₃)—(CH₂)_(n) ¹⁸F, O—(CH₂)_(n)F, O—(CH₂)_(n) ¹⁸F, CONH(CH₂)_(n)F orCONH(CH₂)_(n) ¹⁸F (n being in each case 2 to 4) and R₃ is hydroxy,(C1-4)alkoxy, hydrogen or nitro, in free base or acid addition saltform.
 2. A process for the production of a compound of formula I asdefined in claim 1 and its acid addition salts, comprising the steps ofa) for the production of a compound of formula I which contains no ¹¹Cor ¹⁸F atom, reacting a compound of formula II

 wherein X is as defined in claim 1 and Hal is Cl, Br or I, with5-(2-fluoro-ethylamino)thiazolyl-2-boronic acid or a compound of formulaIII

 wherein Y and R₃ are as defined above and R′₂ is a group R₂ as definedabove which contains no ¹¹C or ¹⁸F atom, or b) for the production of acompound of formula I wherein R₁ is 5-(2-¹⁸F-ethylamino)-thiazol-2-yl,reacting a compound of formula I wherein R₁ is5-(2-mesyloxy-ethylamino)thiazol-2-yl or5-(2-tosyloxy-ethylamino)-thiazol-2-yl with ¹⁸FΘ, or c) for theproduction of a compound of formula I wherein R₂ is NH¹¹CH₃, N(CH₃)¹¹CH₃or N(¹¹CH₃)₂, reacting a compound of formula I wherein R₂ is NH₂ orNHCH₃ with ¹¹CH₃I, or d) for the production of a compound of formula Iwherein R₂ is NH(CH₂)_(n) ¹⁸F, N(CH₃)—(CH₂)_(n) ¹⁸F, O—(CH₂)_(n) ¹⁸F orCONH(CH₂)_(n) ¹⁸F, reacting a compound of formula I wherein R₂ is,respectively, NH(CH₂)_(n)OTs or NH(CH₂)_(n)OMs, N(CH₃)—(CH₂)_(n)OTs orN(CH₃)—(CH₂)_(n)—OMs, O—(CH₂)_(n)OTs or O—(CH₂)_(n)—OMs, orCONH(CH₂)_(n)OTs or ONH(CH₂)_(n)OMs, with ¹⁸FΘ, and recovering theresulting compound of formula I in free base form or acid addition saltform.
 3. A composition for labeling histopathological structures invitro or in vivo, comprising a compound of formula I as defined in claim1, in free base or acid addition salt form.
 4. A method for labelinghistopathological structures in vitro or in vivo, comprising contactingbrain tissue with a compound of formula I as defined in claim 1, in freebase or acid addition salt form.
 5. A method according to claim 4, forlabeling β-amyloid deposits.
 6. A method according to claim 4,comprising administering the compound of formula I to a patient.
 7. Amethod according to any of claims 4, comprising the further step ofdetermining whether the compound of formula I labeled the targetstructure.
 8. A method according to claim 7, comprising observing thetarget structure labeled with a non-radioactive compound of formula I,using fluorescence microscopy.
 9. A method according to claim 7,comprising observing the target structure labeled with a radioactivecompound of formula I, using positron emission tomography (PET).
 10. Amethod according to claim 4 for diagnosing Alzheimer's disease.
 11. Amethod according to claim 10, for monitoring the effectiveness of atherapeutic treatment of Alzheimer's disease.
 12. A method according toclaim 4, for detecting histopathological hallmarks of Alzheimer'sdisease.
 13. A package comprising a compound of formula I,

wherein X is O or S, R₁ is 5-(2-fluoro-ethylamino)-thiazol-2-yl,5-(2-¹⁸F-ethylamino)-thiazol-2-yl or a group of formula (a)

wherein Y is CH or N, R₂ is NH₂ or NHCH_(3,) and R₃ is hydroxy,(C1-4)alkoxy, hydrogen or nitro, in free base or acid addition saltform, together with instructions for the production of the compound offormula I wherein R₂ is NH¹¹CH₃, N(CH₃)¹¹CH₃ or N(¹¹CH₃)₂ by reaction ofthe starting material with freshly prepared ¹¹CH₃I.
 14. A packagecomprising as starting material a compound of formula I,

wherein X is O or S, R₁ is 5-(2-fluoro-ethylamino)-thiazol-2-yl,5-(2-¹⁸F-ethylamino)-thiazol-2-yl or a group of formula (a)

 wherein Y is CH or N, R₂ is NH(CH₂)_(n)OTs, NH(CH₂)_(n)OMs,N(CH₃)—(CH₂)_(n)OTs, N(CH₃)—(CH₂)_(n)OMs, O—(CH₂)_(n)OTs,O—(CH₂)_(n)OMs, CONH(CH₂)_(n)OTs or ONH(CH₂)_(n)OMs (n being in eachcase 2 to 4), wherein OMs corresponds to mesylate and OTs to tosylate,and R₃ is hydroxy, (C1-4)alkoxy, hydrogen or nitro, in free base or acidaddition salt form, together with instructions for the production of thecompound of formula I wherein R₂ is NH(CH₂)_(n) ¹⁸F, N(CH₃)—(CH2)_(n)¹⁸F, O—(CH₂)_(n) ¹⁸F or CONH(CH₂)_(n) ¹⁸F by a suitable reaction cascadeof the starting material with ¹⁸FΘ.